In vitro drug metabolism reagent and uses thereof

Abstract

The present disclosure provides an in vitro reagent for evaluating xenobiotic metabolism in a cell culture based assay. The in vitro reagent is an admixture of metabolically competent cells and exogenous drug metabolizing enzyme co-factors follow by cryopreservation in the absence of cryopreservation agent so that the cells would be rendered permeable upon thawing due to plasma membrane disruption (while maintaining the integrity of organelles). The permeabilized plasma membranes allow ready diffusion of the exogenous cofactors into the cells to enhance the activities of cellular drug metabolizing enzymes. Addition of a xenobiotic test compound to the thawed in vitro reagent allows metabolism of the test compound by the metabolically competent cells, with metabolites readily diffusible outside the cells due to the permeabilized plasma membranes.

1 . An in vitro reagent for evaluating xenobiotic metabolism, wherein the reagent is a cryopreserved mixture comprising:
 a) metabolically competent cells wherein cell membranes are permeabilized upon thawing;   b) exogenous drug metabolizing enzyme co-factors, and   c) a buffered solution,   wherein the reagent does not comprise a cryopreservative agent.   
     
     
         2 . The reagent of  claim 1 , wherein the metabolically competent cells are hepatocytes or enterocytes. 
     
     
         3 . (canceled) 
     
     
         4 . The reagent of  claim 1 , wherein the metabolically competent cells are engineered to contain cytochrome P450 isoforms. 
     
     
         5 . The reagent of  claim 1 , wherein the metabolically competent cells comprise drug metabolizing enzyme (DME) activities. 
     
     
         6 - 9 . (canceled) 
     
     
         10 . The reagent of  claim 1 , wherein the drug metabolizing enzyme co-factors are selected from β-Nicotinamide adenine dinucleotide 2′-phosphate (NADPH), Uridine 5′-diphosphoglucuronic acid (UDPGA), 3′-Phosphoadenosine 5′-phosphosulfate (PAPS), N-acetyl coenzyme A, s-adenosyl methionine, amino acids, carnitine, and L-glutathione. 
     
     
         11 . The reagent of  claim 1 , wherein the reagent is stored frozen at a temperature of −10° C. to about −80° C. 
     
     
         12 . The reagent of  claim 11 , wherein the reagent is thawed prior to use for evaluating xenobiotic metabolism. 
     
     
         13 . The reagent of  claim 1 , wherein the reagent is further added to and comprises a mammalian or non-mammalian culture, wherein the reagent provides exogenous metabolic functions for evaluation of a physiological effect of metabolites of a xenobiotic. 
     
     
         14 . The reagent of  claim 13 , wherein the non-mammalian culture comprises  Salmonella  bacteria used in an Ames  Salmonella  histidine reversion assay (Ames test) for genotoxicity. 
     
     
         15 . The reagent of  claim 13 , wherein the mammalian or non-mammalian culture comprises cells used in a genotoxicity assay. 
     
     
         16 . The reagent of  claim 13 , wherein the mammalian or non-mammalian culture comprises cells used in a cytotoxicity assay. 
     
     
         17 . The reagent of  claim 13 , wherein the mammalian or non-mammalian culture comprises cells used in a pharmacological assay. 
     
     
         18 . The reagent of  claim 1 , wherein the reagent is provided in a kit and further comprises instructions for evaluating xenobiotic metabolism using the reagent. 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . A method for manufacturing an in vitro reagent for evaluating xenobiotic metabolism, comprising:
 a) permeabilizing metabolically competent cells;   b) adding exogenous drug metabolizing enzyme co-factors either before or after the metabolically competent cells are permeabilized; and   c) providing a buffered solution,   wherein the reagent does not comprise a cryopreservative agent.   
     
     
         22 . The method of  claim 21 , wherein the reagent is further stored frozen at a temperature of −10° C. to about −80° C. 
     
     
         23 . (canceled) 
     
     
         24 . The method of  claim 21 , wherein the metabolically competent cells are permeabilized by freezing and thawing in the absence of a cryopreservative agent. 
     
     
         25 - 46 . (canceled) 
     
     
         47 . A method for evaluating xenobiotic metabolism, comprising:
 a) providing the cryopreserved in vitro reagent of  claim 1 ;   b) thawing the in vitro reagent and placing in a cell culture vessel;   c) introducing the xenobiotic test compound into the cell culture vessel;   d) incubating the test compound for 0.5 h to 10 days at 33-40° C.; and,   e) performing an end point assay of the in vitro reagent or cell culture medium to determine metabolism of the xenobiotic test compound.   
     
     
         48 - 52 . (canceled) 
     
     
         53 . The method of  claim 47 , wherein the xenobiotic agent is a drug or drug candidate, an industrial chemical, or an environmental pollutant. 
     
     
         54 . (canceled) 
     
     
         55 . (canceled) 
     
     
         56 . The method of  claim 53 , wherein the drug or drug candidate is selected from the group consisting of an organic compound, an inorganic compound, a hormone, a growth factor, a cytokine, a reception, an antibody, an enzyme, a peptide, an aptomer and a vaccine. 
     
     
         57 . The method of  claim 47 , wherein step b) does not further comprise a centrifugation or cell counting step. 
     
     
         58 - 78 . (canceled)